Potential fumigant toxicity of essential oils against Sitotroga cerealella (Olivier) (Lepidoptera: Gelechiidae) and its egg parasitoid Trichogramma evanescens (Hymenoptera: Trichogrammatidae)

Sitotroga cerealella is a serious pest of a wide range of stored cereal grains. An essential element of an integrated pest control approach is the application of plant oils as a substitute for chemical insecticides. This study aimed to investigate the fumigant toxicity of Allium sativum and Mentha piperita essential oils against S. cerealella adult moths and the egg parasitoid Trichogramma evanescens. Gas chromatography–mass spectrometry analyses detected that Diallyl trisulfide (37.97%) and dl-Menthol (47.67%) as main compounds in A. sativum and M. piperita, respectively. The results showed that, A. sativum at 10.0, 5.0, and 2.5 µL/L air resulted in 100% insect mortality after 24 h exposure. The concentrations of 10.0 and 5.0 µL/L air of M. piperita oil resulted in 100 and 96% insect mortality, respectively. The parasitoid adult emergence in the F1 reduced when exposed to LC99 of A. sativum and M. piperita oils by 10.89 and 9.67%, respectively. Also, the parasitism of emerged parasitoid decreased by 9.25 and 5.84% (class I-harmless), respectively. Therefore A. sativum and M. piperita have the potential to be used as bio-fumigant for the management of S. cerealella and can be used alongside the T. evanescens in integrated pest management.


Potential fumigant toxicity of essential oils against
Sitotroga cerealella is a serious pest of a wide range of stored cereal grains.An essential element of an integrated pest control approach is the application of plant oils as a substitute for chemical insecticides.This study aimed to investigate the fumigant toxicity of Allium sativum and Mentha piperita essential oils against S. cerealella adult moths and the egg parasitoid Trichogramma evanescens.Gas chromatography-mass spectrometry analyses detected that Diallyl trisulfide (37.97%) and dl-Menthol (47.67%) as main compounds in A. sativum and M. piperita, respectively.The results showed that, A. sativum at 10.0, 5.0, and 2.5 µL/L air resulted in 100% insect mortality after 24 h exposure.The concentrations of 10.0 and 5.0 µL/L air of M. piperita oil resulted in 100 and 96% insect mortality, respectively.The parasitoid adult emergence in the F1 reduced when exposed to LC 99 of A. sativum and M. piperita oils by 10.89 and 9.67%, respectively.Also, the parasitism of emerged parasitoid decreased by 9.25 and 5.84% (class I-harmless), respectively.Therefore A. sativum and M. piperita have the potential to be used as bio-fumigant for the management of S. cerealella and can be used alongside the T. evanescens in integrated pest management.
Most of the world's population depends on cereals as a source of carbs, vitamins, minerals, fiber, oils, fats, and protein 1 .Therefore, the explosion of the human population in many developing countries led to an increase in the global demand for major production of cereal crops 2 .Wheat, Triticum sp., is a widespread crop that is grown in various parts of the world and can be attacked by a plethora of pests 3 .Beetles and moths are among the insect pests that severely harm wheat crops 4 .The Angoumois grain moth Sitotroga cerealella (Olivier) (Lepidoptera: Gelechiidae) is one of the most significant pests of cereal grains such as wheat, corn, barley, and rice throughout the world.The pest's larvae cause injury to wheat grains by feeding on seed contents, which reduces the weight and nutritional value of the grains and makes them more susceptible to diseases.Synthetic chemicals are commonly used to manage S. cerealella; however, indiscriminate chemical treatment has a number of negative impacts including increased pest resistance and the spread of secondary pests, as well as negative effects on the environment and human health 5 .As a result, the employment of alternative control techniques has become essential.Finding efficient and eco-friendly alternatives to chemical pesticides presents a significant challenge in limiting the harm wrought by the S. cerealella pest and reducing crop losses.Egg parasitoids have been used in biological control far more frequently than other natural enemies, especially those of the genus Trichogramma (Hymenoptera: Trichogrammatidae).Also, many research studies recently described plant essential oils as ecofriendly agents to control various pests with the least possible effects on the environment and human health.In order to suppress the population of S. cerealella in post-harvest storage, the use of essential oil has been examined as an alternative viable technique for managing this pest.The use of plant essential oils as safe, biodegradable insecticides has gained popularity as a viable substitute for dangerous fumigant pesticides in the fight against stored-product insect pests.Fumigation is a useful technique for controlling pests in stored goods.As a

Essential oils and their phytochemical screening
Essential oils of A. sativum and M. piperita were obtained from National Research Centre (NRC), Cairo, Egypt.For Gas Chromatography-Mass Spectrometry (GC-MS) analysis, the samples were dissolved in chloroform at the ratio of 50 µL oil:1 mL chloroform and injected into GC.The GC-MS system (Agilent Technologies) was equipped with gas chromatograph (7890B) and mass spectrometer detector (5977A) at NRC.The GC was equipped with HP-5MS column (30 m × 0.25 mm internal diameter and 0.25 μm film thickness).The carrier gas was helium, which was used at a flow rate of 3.0 mL/min at a split ratio (1:10), an injection volume of 1 µL, and the temperature programme as follows: 40 ºC for 1 min; 10 ºC per minute to 200 ºC and held for 1 min; 20 ºC per minute to 220 ºC and held for 1 min; 30 ºC per minute to 320 ºC and held for 3 min.The injector and detector were kept at 250 ºC and 320 ºC, respectively.By using a spectral range of m/z 30-550 and a solvent delay of 2.5 min, mass spectra were produced using electron ionization (EI) at 70 eV.The quad was 150 ºC warmer than the mass temperature of 230 ºC.By comparing the spectrum fragmentation pattern with those found in the Wiley and NIST Mass Spectral Library data, it was possible to identify various constituents of A. sativum and M. piperita oils.

Sitotroga cerealella
The S. cerealella moths were obtained from naturally infested grains stored in a local warehouse in Cairo governorate, Egypt.The stock culture was maintained for more than 5 generations without exposure to insecticides.The adult moths were reared on intact hard wheat grains inside 1000 mL jars.The resultant moths were collected in special cylinders coated with a wire screen with fine pores that allowed the eggs to pass through and prevent the escape of any moths.In the experiments, the newly emerged adults and freshly deposited eggs were used.Wheat grains were purchased from a local market.In order to kill any insects or parasites present, grains were first stored for about 5 days at − 18 °C before being used.The grains were then adjusted for an additional week before use to the laboratory conditions where the culture was kept.Rearing insects and experiments were carried out under controlled conditions of 28 ± 2 °C, 65 ± 5% RH, and 16:8 (L:D) photoperiod.

Trichogramma evanescens
The egg parasitoid T. evanescens were obtained from the mass rearing of the Laboratory of Biological Control of Insects, Plant Protection Research Institute, Agricultural Research Center, Giza, Egypt.Fresh S. cerealella eggs were glued to paper strips (3 × 8 cm).The strips holding S. cerealella eggs were exposed to T. evanescens adults in glass jars (1000 mL) covered with muslin cloth and tightly tied by a rubber band.Egg strips were renewed daily, and old parasitized eggs were incubated under laboratory conditions.The culture of parasitoid was reared under the previous conditions.

Sitotroga cerealella
The fumigant toxicity of A. sativum and M. piperita essential oils was examined using S. cerealella moth adult stage.Glass jars of 1000 mL capacity provided with their screw lids were used as exposure chambers.Concentrations were carried out by releasing the required amounts of the tested oil (10.0, 5.0, 2.5, 1.25, 0.625 µL/L air) from an automatic micropipette onto Whatman No.1 filter paper disks (2 cm diameter) and were glued on the underside of the screwcap of the glass jar.Ten adults of S. cerealella (1-3 days old) were released into each jar supplied with 40 g of wheat before the lid was closed tightly and the cover was sealed with parafilm.For control group, the insects were maintained without oil exposure (0 µL/L air).Five replicates were used for each treatment.Insect mortality was checked after 24 h to calculate lethal concentration values of LC 99 , LC 50 , and LC 25 for the tested oils.

Trichogramma evanescens
Eemergence rate of Trichogramma evanescens.To evaluate the fumigant toxicity of A. sativum and M. piperita essential oils on the emergence rate of the adult parasitoid, an equal number of 1-day-old S. cerealella eggs (110 ± 10) for each treatment were dispersed on self-adhesive strip (1 mm diameter) and exposed to adult parasitoid for 24 h.After 4 days the eggs became black (i.e., clear evidence of parasitism), then the parasitized egg strips were kept in glass jars.Egg strips were exposed to the previously estimated LCs of essential oils vapor on S. cerealella (LC 99 , LC 50 , and LC 25 ) for 24 h as described previously.After exposure, the egg strips were placed separately in glass test tubes (Fig. 1), and the number of emergence holes (Fig. 2a) were counted to evaluate the percentage of adult emergence.
Parasitism rate of emerged Trichogramma evanescens (F1).This experiment was performed to estimate the efficiency of emerged T. evanescens adults (F1) of previous experiment.Freshly mated parasitoid (ten adults/each egg strip) were released in glass jars (1000 mL) containing S. cerealella eggs strips (110 ± 10).After 24 h of exposure to the parasitoids, S. cerealella egg masses were removed from the jars and put into individual transparent test tubes.The number of darkened eggs was counted under binocular to determine the parasitism% (Fig. 2b).As a control, the individuals were maintained in test tubes without any fumes.For each treatment, the studies were repeated ten times.
The proportion reduced emergence and parasitism rates were computed as 12 :

Data analysis
The mortality, emergence rate, and parasitism rate results were analyzed using One-Way ANOVA, and Duncan's test was used to determine whether there were significant differences between the treatments (P < 0.05).Average mortality data for each treatment were subjected to probit analysis 13 to obtain the lethal concentration (LC) values.LC values were considered significantly different at the P < 0.05 level.Data analyses were performed using SPSS version 18.0 14 .
(1) 100 − mean %mean of the treatment %mean of the control * 100

Toxicity study
Fumigant toxicity of A. sativum and M. piperita oils against S. cerealella adult moths is illustrated in Fig. 4. The mortality rate increased as the oil concentration increased after a 24-h exposure.The highest percentage of insect mortality (100%) was observed for A. sativum oil concentrations of 10.0, 5.0, and 2.5 µL/L air.The mortality rate decreased to 80% following 1.25 µL/L air A. sativum fumigation.In contrast, only 6.0% of insects died when A. sativum was applied at the lowest concentration (0.625 µL/L air).Comparably, higher M. piperita oil concentrations (10.0, 5.0 µL/L air) led to a higher mortality rate of 96% and 100%, respectively.Also, 2.5 and 1.25 µL/L air caused mortality percentage > 50% after 24 h exposure (Fig. 4).Toxicity data obtained in Table 3 indicated that LC 25 , LC 50 , and LC 99 of A. sativum oil against S. cerealella adult moths after 24 h exposure was 0.82, 1.04, and 1.80 µL/L air, respectively.The LC values resulted after fumigation with M. piperita oil were 0.79, 1.88, and 5.65 µL/L air for LC 25 , LC 50 , and LC 99 , respectively.

Effect of A. sativum and M. piperita essential oils on the efficacy of T. evanescens
Results shown in Table 4 demonstrated that the exposure of parasitized eggs of S. cerealella to the fumes of the tested oils affected the emergence rate of the parasitoid.The highest emergence rates of T. evanescens were 92.51%   and 93.22% at treatment of LC 25 A. sativum and M. piperita, respectively.Furthermore, the adult emergence reduced when parasitized S. cerealella eggs were exposed to the highest tested concentration LC 99 of A. sativum and M. piperita oils by 10.89 and 9.67% (class I-harmless).In the same line, the parasitism rate of the emerged female of F1 generation was affected by A. sativum and M. piperita (F = 49.042,P < 0.001and F = 44.54,P < 0.001) respectively.When the fresh S. cerealella eggs were offered to the emerged F1 females after LC 99 treatment, the parasitism was decreased by 9.25 and 5.84% (class I), respectively, and these oils were therefore classified as harmless (Table 4).

Discussion
Plant based insecticides such as essential oils are considered as a promising component for pest control and an alternative to synthetic chemicals.The immediate or long-term implications of insecticides on natural enemies should be considered when integrating synthetic or bio-insecticides with biological control agents to control major agricultural pests 15 .In the current work, the essential oils of A. sativum and M. piperita effectively caused mortality of S. cerealella adult stages.It was observed that the toxicity increased with increasing oil concentration.Earlier studies have mentioned that different essential oils showed an insecticidal impact on Sitotroga cerealella (Olivier) (Lepidoptera: Gelechiidae) and its egg parasitoid Trichogramma evanescens (Hymenoptera: Trichogrammatidae) Huda H. Elbehery * & Samar S. Ibrahim

Figure 1 .
Figure 1.The parasitized Sitotroga cerealella eggs strip (1 mm diameter) kept separately inside the glass tubes closed with cotton plug.

Figure 4 .
Figure 4. Mortality of Sitotroga cerealella adult moths after 24 h exposure to A. sativum or M. piperita oil.Mean (± SE) values with different letters within the same oil treatment are significantly different (P < 0.05) (ANOVA) (Duncan test).

Table 1 .
GC-MS analysis of A. sativum.
a RT: retention time.b Area: area of the peak.c Area sum: peak area/ total peak area.

Table 2 .
GC-MS analysis of M. piperita.a RT: retention time.b Area: area of the peak.c Area sum: peak area/ total peak area.

Table 3 .
Toxicity of A. sativum and M. piperita oil on Sitotroga cerealella adult moths after 24 h exposure.

Table 4 .
Emergence rate, parasitism rate, reduction (E, P%), and toxic class (TC) of Trichogramma evanescens from Sitotroga cerealella eggs.Means followed by the same letter per column do not differ by Duncan test (P < 0.05).TC = toxic class, E% = emergence reduction, P% = parasitism reduction.